Neurogenesis Plays A Role In The Central Diabetes Insipidus Induced By Hypophysectomized Rats

BackgroundMagnocellular neuron both in Paraventricular hypothalamic Nucleus (PVN) and Supraoptic Nucleus (SON) project axons to posterior lobe of the pituitary gland, which form the hypothalamic neurohypophysial tract in mammals. The hypothalamic neurohypophysial tract mainly carry the function of synthesis, transport and release of Arginine Vasporessin (AVP) and oxytocin (OT).Moreover the lack of arginine vasopressin appears increased urine output, decreased urine specific gravity, polydipsia and other symptoms. Pituitary damage of the HNS in rats can be generally modeled by resection of pituitary, the parapharyngeal approach and the internal acoustic approach are the commonly used hypophysectomy operation approach, however, due to relatively heavily in animal injury,complex in postoperative complications,and low in survival rate, parapharyngeal approach is not suggested suitable for long-term observation, and the stereotactic via acoustic approach for modeling is relatively convenient, well postoperative recovery, high survival rate method, but still has exhibit low rate of complete resection of pituitary and molding, also have a limitation on the size of animal, in order to stabilize the molding, exploring for the suitable size of animal is urgenting. Hypophysectomy inducing a series of complex pathophysiological changes still need continuous exploration,many scholars believe that injury of the system hypothalamic pituitary (HNS), resulting in retrograde degeneration of hypothalamic magnocellular neurons, eventually reduce in hormone secretion, which bring about central diabetes insipidus. At the same time, there are many studies suggest that the central nervous system injury may process may be associated with new born cells process, and part of the new cells mature into neurons after differentiation through a period of growth, and exhibit the function of neuron and expression of protein. However, whether neurogenesis exist in PVN or SON in hypophysectomized model, and whether the new cells could be differentiate and mature into neurons releasing hormone are not fully confirmed yet. While the relationship between newborn neurons with central diabetes insipidus are need further elaborated. Clarifing neurogenesis after hypophysectomy, is important to study the neural repair in clinical treatment, also to provide new ideas in clinical. Based on the above considerations, we use the stereotactic instrument for rat pituitary resection, and describe central diabetes insipidus pattern in the postoperative rats to find out a suitable observation time points, meanwhile using the new cell labeling method, tracking postoperative new cells to comfirm existence in PVN and SON, and the counting new cells, to investigate the pattern of neurogenesis in hypophysectomized rats.Objectives:1. Using of stereotactic hypophysectomy instrument in different weight rats, in order to establish stable model in the rat, and observe the characteristic of central diabetes insipidus after surgery, to find suitable observation time window for neurogenesis;2. To confirm whether neurogenesis emerge in PVN or SON of hypophysectomizd rat, meanwhile observe its maturation and survival pattern;Materials and methodsFirst part:Observation of central diabetes insipidus in hypophysectomized rat1. Modeling:using stereotaxic apparatus for rat pituitary resection.2. Experimental groups:experiment one:a total of 60 male SD rats,15 rats in weight150-200g,15 in 200-250g,15 in 250-300g,15 in 300-450g.Experiment two:150-250g rats12,10 rats in the sham group;3. Figure out the CDI incident in hypophysectomied rats in different groups;4. Calculate the short and long survive rate after operation in different groups;5. To observe whether there are remnants in pituitary fossa after surgery, stereotactic was analyzed under the total resection rate;6. Determination of the biological characteristic changes of rat central diabetes insipidus after surgery:measurement of daily urine volume, urine specific gravity, water intake and draw a diabetes insipidus curve;7. Data analysis:using SPSS20.0 software to analysis. Including chi square test, one-way analysis of variance, repeated measurement analysis of variance and each time point of single effect comparative (LSD method), represented by P<0.05 had significant difference.second part:Observing newborn AVP neurons in postoperative rat in different period1. Modeling and grouping:model consistent with the first part, grouping:18 male SD rats, were randomly divided into sham operation group of 6 rats,6 rats in group 10 days after operation,6 in postoperative day 20 group.2. BrdU labelling:BrdU are administrated by intraperitoneal injection, 100mg/kg once a day after surgery, consecutive 7 days injection required.3. Brains sample collection and immunofluorescence analysis:sham operative group and the group of 10 days perfusion after 10th days,20 days group and were sacrificed in 20th day after operation. Perfusing with saline and frozen 4% paraformaldehyde, put out brain and soak in sucrose for gradient dehydration,then frozening embed with embedding agent,finally coronal slices are collected and keep in 4 temperature for used. BrdU staining:After PBS rinse, slices were soak in hydrochloric acid for DNA degeneration and then sections are moved in borate buffer for neutralization,after rinse and incubated with 5%BSA 2 hours later, incubated with mouse anti BrdU antibody (1:800) at 4℃ overnight, next day rinsing with PBS and incubation with fluorescent anti mouse 488 avoiding light, then after dark incubation of DAPI and mounting slices, finally observed under the fluorescent fluorescence microscopy. BrdU/AVP co-immunostaining:after PBS rinsing and incubation with 5%BSA 2hours, then incubated with Rabbit anti AVP antibody and 1:2000 at 4 degrees overnight, and PBS rinsing and incubated with fluorescent second anti rabbit 546 in dark incubation, remain procedure keep in dark conduction. In cubation with DAPI and fixation with methanol, PBS rinse then hydrochloric acid and borate buffer neutralization for DNA degeneration. Then incubation with mouse anti BrdU antibody 1:800 at 4 degrees overnight, after rinsing and with fluorescent mouse anti 488 dark incubation, observed under the fluorescent microscope.4. photographs are captured in fluorescence microscope and count the number of BrdU positive cells and AVP positive cell, number BrdU and AVP dual-positive expression cells.5. Data analysis:using SPSS20.0 software to analysis. Including chi square test, one-way analysis of variance,, represented by P<0.05 had significant difference.Result1. Stereotactic hypophysectomy,:none of the gourp exhibit a predominant postoperative short-term and long-term survival rate, and the full resection rate and diabetes insipidus appeared has no significant difference between the rates.2. The central diabetes insipidus after hypophysectomy in rat,can be divided into 3 stage characterized with the urine volume, water in take and urine specific gravity.3. New cells occurred in the rat supraoptic nucleus and paraventricular nucleus after postoperation.4. New cell nucleus exhibit differentiation and maturation for expression of AVP neurons in the supraoptic nucleus and PVN in 2 seperated time, the new born AVP neurons accounted for the total AVP ratio in postoperations 20 day higher than in that after 10 daysConclusion1. stereotactic instrument aid hypophysectomy is suitable for wide range weight rat;2. the CDI can be successfully modeled by stereotactic hypophysectomy, and urine volume, urine specific gravity and water intake display a three-phase diabetes insipidus pattern in post-operative rats;3. Newborn cells emerge in SON and PVN in hypophysectomized rats, and there is no significant difference in intermittent period and duration;4. After a period of growth new born cell can differentiate into mature neurons expressing AVP, suggesting that neurogenesis may be a additional compensatory response to HNS injury.